Recovery of isoflavones from soy molasses

ABSTRACT

A health supplement composition is disclosed. The health supplement composition contains a solid health supplement material that is separated from a soy molasses material. The solid health supplement material contains at least least two isoflavones.

[0001] This application is a divisional of patent application Ser. No.10/016,461 filed on Oct. 19, 2001 which is a divisonal application ofpatent application Ser. No. 09/204,808 filed on Dec. 3. 1998, now U.S.Pat. No. 6,323,018, which is a continuation of patent application Ser.No. 09/086,658 filed on May 29, 1998, now U.S Pat. No. 5,919,921, whichis a continuation-in-part of patent application Ser. No. 08/661,845filed on Jun. 11, 1996, now U.S. Pat. No. 5,821,361.

FIELD OF THE INVENTION

[0002] The present invention relates to a health supplement containingat least two isoflavones that is derived from a soy molasses material.

BACKGROUND OF THE INVENTION

[0003] Isoflavones occur in a variety of leguminous plants, includingvegetable protein materials such as soybeans. These compounds includedaidzin, 6″-OAc daidzin, 6″-OMal daidzin, daidzein, genistin, 6″-OAcgenistin, 6″-OMal genistin, genistein, glycitin, 6″-OAc-glycitin,6″-OMal glycitin, glycitein, biochanin A, formononentin, and coumestrol.Typically these compounds are associated with the inherent, bitterflavor of soybeans.

[0004] The isoflavones in soybean materials include isoflavoneglucosides (glucones), isoflavone conjugates and aglucone isoflavones.Isoflavone glucosides have a glucose molecule attached to an isoflavonemoiety. Isoflavone conjugates have additional moieties attached to theglucose molecule of an isoflavone glucoside, for example, 6″-OAcgenistin contains an acetate group attached to the six position of theglucose molecule of genistin. Aglucone isoflavones consist solely of anisoflavone moiety.

[0005] Soy contains three “families” of isoflavone compounds havingcorresponding glucoside, conjugate, and aglucone members: the genisteinfamily, the daidzein family, and the glycitein family. The genisteinfamily includes the glucoside genistin; the conjugates 6″-OMal genistin(6″-malonate ester of genistin) and 6″-OAc genistin (6″-acetate ester ofgenistin); and the aglucone genistein. The daidzein family includes theglucoside daidzin; the conjugates 6″-OMal daidzin and 6″-OAc daidzin;and the aglucone daidzein. The glycitein family includes the glucosideglycitin; the conjugate 6″-OMal glycitin; and the aglucone glycitein.

[0006] In the production of commercial products, such as vegetableprotein concentrates, the focus has been to remove these materials. Forexample, in a conventional process for the production of a soy proteinconcentrate in which soy flakes are extracted with an aqueous acid or anaqueous alcohol to remove water soluble materials from the soy flakes,much of the isoflavones are solubilized in the extract. The extract ofwater soluble materials, including the isoflavones, is soy molasses. Thesoy molasses is a byproduct material in the production of soy proteinconcentrate which is typically discarded. Soy molasses, therefore, is aninexpensive and desirable source of isoflavones, provided that theisoflavones can be separated from the soy molasses.

[0007] It has recently been recognized that the isoflavones contained invegetable protein materials such as soybeans have medicinal value. Theaglucone isoflavones are of particular interest. Genistein and daidzeinmay significantly reduce cardiovascular risk factors. “Plant andMammalian Estrogen Effects on Plasma Lipids of Female Monkeys”,Circulation, vol. 90, p. 1259 (October 1994). Genistein and daidzein arealso thought to reduce the symptoms of conditions caused by reduced oraltered levels of endogenous estrogen in women, such as menopause orpremenstrual syndrome. Further, it has recently been recognized thataglucone isoflavones may inhibit the growth of human cancer cells, suchas breast cancer cells and prostate cancer cells, as described in thefollowing articles: “Genistein Inhibition of the Growth of Human BreastCancer Cells, Independence from Estrogen Receptors and the Multi-DrugResistance Gene” by Peterson and Barnes, Biochemical and BiophysicalResearch, Communications, Vol. 179, No. 1, pp. 661-667, Aug. 30, 1991;“Genistein and Biochanin A Inhibit the Growth of Human Prostrate CancerCells but not Epidermal Growth Factor Receptor TyrosineAutophosphorylation” by Peterson and Barnes, The Prostate, Vol. 22, pp.335-345 (1993); and “Soybeans Inhibit Mammary Tumors in Models of BreastCancer” by Barnes, et al., Mutagens and Carcinogens in the Diet, pp.239-253 (1990).

[0008] The aglucone isoflavones have the following general formula:

[0009] wherein, R₁, R₂, R₃ and R₄ may be selected from the groupconsisting of H, OH and OCH₃.

[0010] Genistein has the formula above where R₁═OH, R₂═H, R₃═OH, andR₄═OH, daidzein has the formula above where R₁═OH, R₂═H, R₃═H, andR₄═OH, and glycitein has the formula above where R₁═OH, R₂═OCH₃, R₃═H,and R₄═OH.

[0011] It is therefore to the isoflavones and to the recovery of anisoflavone enriched material from soy molasses to which the presentinvention is directed. The present invention is further directed toisoflavone glucosides and aglucone isoflavones—to the conversion ofisoflavones of soy molasses to isoflavone glucosides and agluconeisoflavones, and to the recovery of an isoflavone glucoside enrichedmaterial and an aglucone isoflavone enriched material from soy molasses.

[0012] A general process for converting vegetable protein isoflavoneconjugates to aglucone isoflavones is known, and is provided in thecurrently pending application U.S. Ser. No. 08/477,102 filed Jun. 7,1995 owned by the assignee of the present application.

[0013] Other processes are known in the art for converting isoflavoneglucosides to aglucone isoflavones, such as described in Japanese PatentApplication 258,669 to Obata, et al. Such processes do not provide forthe recovery of an isoflavone enriched material from soy molasses. Suchprocesses also do not provide for the conversion of isoflavoneconjugates to isoflavone glucosides or to aglucone isoflavones.Furthermore, these processes achieve only a moderate extent ofconversion of isoflavone glucosides to aglucone isoflavones, and requirea substantial period of time to effect this moderate extent conversion.

[0014] It is therefore an object of the present invention to provide anisoflavone enriched material and a process for producing the same fromsoy molasses.

[0015] It is a further object of the present invention to provide anisoflavone glucoside enriched material and a process for producing thesame from soy molasses.

[0016] It is still a further object of the present invention to providean aglucone isoflavone enriched material and a process for producing thesame from soy molasses.

SUMMARY OF THE INVENTION

[0017] The present invention is an isoflavone enriched material and aprocess for recovering the same from a soy molasses material containingisoflavones. The method comprises providing a soy molasses materialcontaining isoflavones, and separating a cake from the soy molassesmaterial at a pH and a temperature sufficient to cause a majority of theisoflavones to be contained in the cake. Preferably the pH is about 3.0to about 6.5 and the temperature is about 0° C. to about 35° C. duringthe separation. The cake is an isoflavone enriched material.

[0018] In one embodiment, a glucoside enriched isoflavone material isformed from the cake of isoflavone enriched material. An aqueous slurryis formed of the isoflavone enriched material. The slurry is treated ata temperature of about 2° C. to about 120° C. and a pH of about 6 toabout 13.5 for a time sufficient to convert isoflavone conjugates in theisoflavone enriched material to isoflavone glucosides. A cake ofisoflavone glucoside enriched material may then be separated from theslurry.

[0019] In another embodiment, an aglucone isoflavone enriched materialis formed from the cake of isoflavone enriched material. An aqueousslurry is formed of the isoflavone enriched material. The slurry istreated at a temperature of about 2° C. to about 120° C. and a pH ofabout 6 to about 13.5 for a time sufficient to convert isoflavoneconjugates in the isoflavone enriched material to isoflavone glucosides.An enzyme capable of cleaving 1,4-glucoside bonds is contacted with theisoflavone glucosides in the slurry at a temperature of about 5° C. toabout 75° C. and a pH of about 3 to about 9 for a time sufficient toconvert the isoflavone glucosides to aglucone isoflavones. A cake ofaglucone isoflavone enriched material may be separated from the slurry.

[0020] In another aspect, the present invention is an isoflavoneglucoside enriched material and a process for recovering the same fromsoy molasses. The soy molasses is treated at a temperature of from about2° C. to about 120° C. and at a pH value of between about 6 to about13.5 for a time period sufficient to convert isoflavone conjugatescontained in the soy molasses to isoflavone glucosides. A cake ofisoflavone glucoside enriched material is separated from the soymolasses material at a pH and a temperature sufficient to cause amajority of the isoflavone glucosides to be contained in the cake.

[0021] In another aspect, the present invention is an agluconeisoflavone enriched material, and a process for recovering the same fromsoy molasses. The soy molasses is treated at a temperature of from about2° C. to about 120° C. and at a pH value of between about 6 to about13.5 for a time period sufficient to convert isoflavone conjugatescontained in the soy molasses to isoflavone glucosides. An enzymecapable of cleaving 1,4-glucoside bonds is contacted with the isoflavoneglucosides in the soy molasses material at a temperature of about 5° C.to about 75° C. and a pH of about 3 to about 9 for a time periodsufficient to convert the isoflavone glucosides to aglucone isoflavones.A cake of aglucone isoflavone enriched material is separated from thesoy molasses material at a pH and a temperature sufficient to cause amajority of the aglucone isoflavones to be contained in the cake.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0022] The starting material of a process of the present invention maybe soy molasses. Soy molasses is generally considered to be the soysolubles removed from soy insolubles by washing with alcohol or aqueousacid. Soy solubles included in the soy molasses are primarilycarbohydrates, highly soluble protein, and isoflavones. Soy insolublesnot included in the soy molasses include vegetable fiber materials,insoluble soy protein, cellulose, and insoluble hemicellulose. Soymolasses is a by-product of many commercial processes involving soybeansor soybean derivatives, such as processes for producing proteinconcentrate products. Accordingly, soy molasses is produced in largequantities to such an extent that soy molasses is a relativelyinexpensive commercially available commodity.

[0023] Alternatively, the starting material for a process of the presentinvention may be a soy material which contains isoflavones andcarbohydrates from which a soy molasses may be formed. Such soymaterials include, but are not limited to, soy meal, soy flour, soygrits, soy flakes, and mixtures thereof, which preferably have beenchemically or mechanically defatted.

[0024] The starting soy material is extracted or washed with anextractant effective to remove substantial amounts of the isoflavonesand carbohydrates present in the soy material. After the extraction orwash, the resulting extract is separated from the insoluble residual soymaterials to form a soy molasses. The extractant preferably is selectedfrom an aqueous low molecular weight alcohol solution, or an aqueoussolution having a pH at about the isoelectric point of soy protein. Forexample, preferred aqueous alcohols include aqueous methanol, aqueousethanol, aqueous isopropyl alcohol, and aqueous propanol, where thealcohol to water ratio of the solution is preferably at least 40:60, andmore preferably is at least 60:40, and most preferably falls within arange of from 65:35 to 90:10. A preferred aqueous solution having a pHat about the isoelectric point of soy protein is water adjusted with asuitable acid, preferably a mineral acid such as hydrocholoric acid orsulfuric acid, to a pH of from about 3.5 to about 5.5, more preferablyto a pH of from about 4 to about 5, and most preferably to a pH of fromabout 4.4 to about 4.6.

[0025] In a preferred embodiment, the soy molasses is produced fromdefatted soy flakes from which oil has been removed by solventextraction in a conventional manner. The defatted soy flakes areextracted with water which has been adjusted to an acidic pH, preferablyabout pH 4 to about pH 5, by the addition of one or more suitable acidssuch as acetic acid, sulfuric acid, phosphoric acid, hydrochloric acidor any other suitable reagent. Preferably the ratio of the acidicextractant to soy flakes is about 10:1 to about 20:1 by weight, and morepreferably from about 12:1 to about 16:1. To improve the efficacy of theextraction, the temperature of the extractant may be elevated above roomtemperature, preferably between about 32° C. and about 55° C. After theextraction, the soy molasses is removed from the soy insolubles.

[0026] In another embodiment, defatted soy flakes are extracted withaqueous alcohol to produce the soy molasses. Preferably, the flakes areextracted with about 80% aqueous ethanol at a ratio of about 10:1 toabout 20:1 by weight of extractant to soy flakes, and more preferablyfrom about 12:1 to about 16:1 by weight of extractant to soy flakes. Thetemperature of the alcohol extractant may be elevated above roomtemperature, preferably from about 32° C. to about 55° C., to improvethe efficacy of the extraction. The soy molasses is then removed fromthe soy insolubles

[0027] The soy molasses is then condensed. The soy molasses may becondensed by conventional methods for removing a liquid, including, butnot limited to, evaporation, preferably under reduced pressure, steamstripping, or distillation. The condensed soy molasses may contain atleast 10% solids, by weight, and preferably contains from about 25% toabout 60% solids, by weight, and more preferably contains from about 30%to about 50% solids, by weight. For example, condensed soy molasses istypically an aqueous mixture containing about 50% or more solids thatcomprise about 6% (based on the total weight of the soy molasses)protein, about 3% ash, about 5% fat, and about 36% carbohydrates. “Soymolasses material” as that term is used herein refers to a compositioncontaining soy molasses, a condensed soy molasses, and/or derivatives ofsoy molasses such as an isoflavone glucoside enriched soy molassesmaterial and an aglucone isoflavone enriched soy molasses material.Accordingly, these terms are used interchangeably herein.

[0028] An isoflavone enriched material may be recovered from thecondensed soy molasses material. The condensed soy molasses material maybe diluted with water to a solids content of from about 6% to about 13%,with 13% being most preferred. Dilution of the condensed soy molassesmaterial is not a requirement for the process, however, diluting therelatively thick condensed soy molasses material facilitates processingthe material.

[0029] The condensed soy molasses material, preferably diluted, istreated at a pH and a temperature at which a majority of the isoflavoneswill separate from the liquid fraction of the condensed soy molassesupon performing a separation procedure. In a preferred embodiment, thecondensed soy molasses material is treated at a pH of about 3.0 to about6.5 and a temperature of about 0° C. to about 35° C. to maximize theinsolubility of the isoflavones in the liquid fraction of the condensedsoy molasses. Isoflavones may be separated from the liquid fraction ofthe condensed soy molasses at pH values outside the preferred range andat temperatures above 35° C., however, these conditions are lesspreferred since less isoflavones are separated in the separationprocedure. The pH of the condensed soy molasses may be adjusted with asuitable conventional acidic or basic reagent, if necessary. It is mostpreferred that the pH of the condensed soy molasses be adjusted to about4.5. It is also preferred to chill or cool the condensed soy molasses toa temperature of about 0° C. to about 10° C., and most preferably to atemperature of about 4° C. to about 7° C.

[0030] The condensed soy molasses material is then subjected to aseparation procedure to separate a cake of isoflavone enriched materialfrom the liquid fraction of the condensed soy molasses material. Theseparation is performed while the soy molasses material is maintainedunder the previously described pH and temperature conditions.

[0031] In one embodiment, the cake of isoflavone enriched material isseparated by centrifuging the soy molasses material and decanting thesupernatant from the cake. Centrifugation is preferably performed atabout 3,000 to about 10,000 rpm for approximately 30 minutes at about 0°C. to about 10° C.

[0032] In another embodiment, the isoflavone enriched cake may beseparated from the soy molasses material by filtration. Preferably thefiltration is done at the previously described pH and temperatureconditions, most preferably at a pH of about 4.5 and a temperature ofabout 0° C. to about 10° C.

[0033] The separated cake of isoflavone enriched material contains asignificant amount of isoflavones therein. Preferably the cake ofisoflavone enriched material contains at least 20 mg of isoflavones pergram of cake material, on a dry basis (at least about 2% isoflavones byweight) and more preferably contains from about 20 mg to about 50 mg ofisoflavones per gram of cake material (from about 2% to about 5%isoflavones, by weight).

[0034] In another aspect of the present invention, a cake of isoflavoneglucoside enriched material may be recovered from soy molasses. A soymolasses material is obtained as described above. Although not arequirement, it is preferred that the soy molasses material be acondensed soy molasses material diluted to a solids content of fromabout 6% to about 13%, and most preferably about 13%, to facilitateprocessing the material.

[0035] A conversion operation is then performed on the soy molassesmaterial to convert isoflavone conjugates in the soy molasses materialto isoflavone glucosides. A substantial portion of the isoflavones inthe soy molasses material are isoflavone conjugates, therefore theconversion substantially increases the amount of isoflavone glucosidesin the soy molasses material. The conversion has been found to bedependent on the pH and the temperature of the soy molasses material.

[0036] The pH range for conversion of the isoflavone conjugates toisoflavone glucosides in a soy molasses material is from about 6 toabout 13.5. The pH of the soy molasses should be adjusted to the desiredpH, if necessary, with a suitable base, caustic agent, or basic reagentif the pH is to be raised, or, if the pH is to be lowered, with asuitable acid or acid reagent. The conversion of isoflavone conjugatesto isoflavone glucosides has been found to be base catalyzed, and so itis most preferred to utilize a high pH to achieve rapid conversion. Themost preferred pH for conversion of the isoflavone conjugates toisoflavone glucosides is a pH of about 9 to about 11.

[0037] The temperature range for conversion of the isoflavone conjugatesto isoflavone glucosides in soy molasses is from about 2° C. to about120° C. The temperature range at which the conversion readily occursdepends on the pH of the soy molasses material. The inventors have foundthat the conversion occurs easily at lower temperatures when the pH isrelatively high. For example, at a pH of about 11 the conversion occursrapidly and efficiently at a temperature range of about 5° C. to about50° C. At a pH of about 9 conversion occurs efficiently within atemperature range of about 45° C. to about 75° C. When the pH of the soymolasses material is relatively low, the conversion occurs at highertemperatures. For example, at a pH of about 6, the conversion occurswithin a temperature range of about 80° C. to about 120° C. In apreferred embodiment, the conversion is effected at about 35° C. and apH of about 11. In another preferred embodiment, the conversion iseffected at a temperature of about 73° C. and a pH of about 9.

[0038] The time period required for conversion of isoflavone conjugatesto isoflavone glucosides depends primarily upon the pH and temperaturerange utilized in the soy molasses material. Such conversion timestypically range from about 15 minutes up to several hours or longer.Conversion occurs more rapidly at a higher pH and at a highertemperature. At a pH of about 9, conversion is substantially complete inabout 4 hours to about 6 hours at 73° C. In a most preferred embodiment,the isoflavone conjugates are converted to isoflavone glucosides inabout 30 minutes to about 1 hour, preferably about 45 minutes, at a pHof about 11 and at a temperature of about 35° C.

[0039] The conversion of the isoflavone conjugates to isoflavoneglucosides is remarkably efficient, converting at least a majority, andpreferably substantially all of the isoflavone conjugates present toisoflavone glucosides. The term a “majority” refers to an extent ofconversion of at least about 50%. The term “substantially all” refers toan extent of conversion of at least about 80%, and most preferably atleast about 90%.

[0040] Following the conversion of the isoflavone conjugates toisoflavone glucosides, a cake of isoflavone glucoside enriched materialmay be separated from the soy molasses material. The soy molasses iscondensed by conventional means, if the soy molasses material is notalready condensed. The condensed soy molasses material is treated at apH and a temperature at which a majority of the isoflavone glucosideswill separate from the liquid fraction of the condensed soy molassesmaterial in a separation procedure. In a preferred embodiment thecondensed soy molasses material is maintained at a pH of about 3 toabout 6.5, most preferably about 4.5, and at a temperature of about 0°C. to about 35° C., preferably about 0° C. to about 10° C., and mostpreferably about 4° C. to about 7° C., during the separation process.The pH of the condensed soy molasses material may be adjusted with asuitable conventional acidic or basic reagent, if necessary.

[0041] The separation may be effected by conventional means forseparating solids from a liquid. The isoflavone glucoside enriched cakeis preferably separated by centrifugation or filtration as describedabove with respect to separating an isoflavone enriched cake from theliquid fraction of a condensed soy molasses material.

[0042] The separated cake of isoflavone glucoside enriched materialcontains a significant amount of isoflavone glucosides therein.Preferably the cake of isoflavone glucoside enriched material containsat least 20 mg of isoflavone glucosides per gram of cake material, on adry basis (at least about 2% isoflavone glucosides by weight) and morepreferably contains from about 20 mg to about 50 mg of isoflavoneglucosides per gram of cake material (from about 2% to about 5%isoflavone glucosides, by weight).

[0043] In yet another aspect of the invention, a cake of agluconeisoflavone enriched material may be recovered from soy molasses. A soymolasses material is obtained as described above, preferably a condensedsoy molasses material diluted with water to a solids content of about 6%to about 13%. The soy molasses material is treated to convert theisoflavone conjugates to isoflavone glucosides as described above.

[0044] An enzymatic conversion operation is then performed on theisoflavone glucoside enriched soy molasses material by contacting asuitable enzyme with isoflavone glucosides in the soy molasses materialat a suitable pH and temperature to convert the isoflavone glucosides toaglucone isoflavones. The two-step conversion process effectivelyconverts substantially all of the isoflavone conjugates and isoflavoneglucosides in the soy molasses material to aglucone isoflavones,substantially increasing the amount of aglucone isoflavones in the soymolasses material.

[0045] The conversion of isoflavone glucosides to aglucone isoflavoneshas been found to be dependent on a variety of factors including thetype of enzymes present in the soy molasses material, distribution ofenzyme concentrations, activities of the enzymes, and the pH andtemperature of the soy molasses material during the conversion. Theenzymes required to effect the conversion are enzymes capable ofcleaving the glucosidic linkage between the isoflavone moiety and theglucose molecule of the isoflavone glucosides. In a preferredembodiment, the enzymes are saccharidase or gluco-amylase enzymescapable of cleaving 1,4-glucoside bonds. The enzymes may be inherentlypresent in the soy molasses material, or may be commercially availableenzymes which are added to the soy molasses material. Inherently presentenzymes are referred to herein as “residual” enzymes, and enzymes thatare added to the soy molasses material are referred to herein as“supplemental” enzymes.

[0046] Sufficient enzyme should be present in the soy molasses materialto convert at least a majority, and preferably substantially all, of theisoflavone glucosides to aglucone isoflavones. Generally, if theresidual enzymes in the soy molasses material are insufficient to effectthe conversion, supplemental enzymes should be added to the soy molassesmaterial. In a preferred embodiment, supplemental enzymes are added tothe soy molasses material regardless of whether sufficient residualenzymes are present in the soy molasses material since addition ofsupplemental enzymes dramatically decreases the time necessary to effectsubstantially complete conversion of the glucosides to aglucones. Ifsupplemental enzymes are added, the supplemental enzymes should be addedso that the total concentration of enzyme present is about 0.1% to about10% by weight of the solids in the soy molasses material on a dry basis.

[0047] Supplemental enzymes are selected based on optimum activity atselected pH and temperature conditions, and cost effectiveness. Thesupplemental enzymes are enzymes capable of cleaving the bond betweenthe isoflavone moiety and the glucose molecule of the isoflavoneglucosides, such as saccharidase and gluco-amylase enzymes capable ofcleaving 1,4-glucoside bonds. Preferred supplemental enzymes arecommercially available alpha-and beta-glucosidase enzymes,beta-galactosidase enzymes, gluco-amylase enzymes, and pectinaseenzymes. Particularly preferred are enzymes such as Biopectinase 100L(which is preferably utilized at a pH range of from about 3 to about 6),Biopectinase 300L (optimum pH range from about 3 to about 6),Biopectinase OK 70L (optimum pH range from about 3 to about 6),Biolactase 30,000 (optimum pH range from about 3 to about 6) NeutralLactase (optimum pH range from about 6 to about 8), all of which areavailable from Quest International, 1833 57th Street, Post Office Box3917, Sarasota, Fla. 34243. Also especially preferred are Lactase F(which is preferably utilized at a pH range of from about 4 to about 6),and Lactase 50,000 (optimum pH range from about 4 to about 6), bothavailable from Amano International Enzyme Co., Inc., Post Office Box1000, Troy, Va. 22974. Other particularly preferred supplemental enzymesinclude G-Zyme G990 (optimum pH from about 4 to about 6) and EnzecoFungal Lactase Concentrate (optimum pH from about 4 to about 6)available from Enzyme Development Corporation, 2 Penn Plaza, Suite 2439,New York, N.Y. 10121; Lactozyme 3000L (which preferably is utilized at apH range from about 6 to about 8), and Alpha-Gal 600L (which preferablyis utilized at a pH range of from about 4 to about 6.5), available fromNovo Nordisk Bioindustrials, Inc., 33 Turner Road, Danbury, Conn. 06813;Maxilact L2000 (which is preferably utilized at a pH range of from about4 to about 6), available from Gist Brocades Food Ingredients, Inc., Kingof Prussia, Pa., 19406; and Neutral Lactase (which is preferablyutilized at a pH range of from about 6 to about 8), available fromPfizer Food Science Group, 205 East 42nd Street, New York, N.Y. 10017.

[0048] The pH range for conversion of the isoflavone glucosides toaglucone isoflavones is from about 3 to about 9. The pH that is utilizeddepends primarily upon the type of enzyme used, and should be selectedaccordingly. The residual enzyme is active within a pH range of about 7to about 9, although it is believed that the pH of the soy molassesmaterial is lowered during the course of the conversion. Thesupplemental enzymes are active within an optimum pH range specified bythe manufacturer of the enzyme, as shown above for several specificenzymes. Typically the supplemental enzymes are active either in aneutral pH range from about 6 to about 8, or in an acidic pH range fromabout 3 to about 6.

[0049] The pH of the soy molasses material may be adjusted to a desiredvalue for conducting the conversion of isoflavone glucosides to agluconeisoflavones. In most instances the pH is reduced from the relativelyhigh or basic pH required to convert the isoflavone conjugates toisoflavone glucosides by the addition of one or more suitable acids suchas acetic acid, sulfuric acid, phosphoric acid, hydrochloric acid, orany other suitable reagent.

[0050] The temperature range of the soy molasses material for theconversion of glucosides to aglucones is from about 5° C. to about 75°C. The temperature significantly affects the activity of the enzymes,and therefore, the rate of conversion. The supplemental enzymes may beactive above 70° C., for example Alpha-Gal 600L is active at 75° C.,however, it is preferred to conduct the conversion at lower temperaturesto avoid enzyme deactivation. In a preferred embodiment, the conversionis effected between about 35° C. and about 45° C.

[0051] The time required for conversion of the glucosides to agluconesdepends upon enzyme-related factors, particularly concentration, and thetemperature and pH of the system. In most instances it is possible toachieve substantially complete conversion within 24 hours, however, itis preferred that supplemental enzyme be added to dramatically increasethe rate of the reaction. The selected supplemental enzyme, enzymeconcentration, pH and temperature preferably cause substantiallycomplete conversion within about 2 hours, and most preferably withinabout 1 hour.

[0052] The conversion of the isoflavone glucosides to agluconeisoflavones is remarkably efficient, converting at least a majority, andpreferably substantially all of the glucosides present to aglucones. Theterm “a majority” refers to an extent of conversion of at least about50%. The term “substantially all” refers to an extent of conversion ofat least about 80%, and most preferably at least about 90%.

[0053] Following the conversion of the isoflavone glucosides to agluconeisoflavones the soy molasses material is condensed as described above,if the soy molasses material has not already been condensed. A cake ofaglucone isoflavone enriched material may be separated from the liquidfraction of the condensed soy molasses material. The soy molassesmaterial is treated at a pH and a temperature at which a majority of theaglucone isoflavones will separate from the liquid fraction of thecondensed soy molasses material in a separation procedure. Preferably,the condensed soy molasses material is maintained at a pH of about 3 toabout 6.5, most preferably about 4.5, and at a temperature of about 0°C. to about 35° C., preferably about 0° C. to about 10° C., and mostpreferably about 4° C. to about 7° C., during the separation process.The pH of the condensed soy molasses material may be adjusted with asuitable conventional acidic or basic reagent, if necessary.

[0054] The separation may be effected by conventional means forseparating solids from a liquid. The aglucone isoflavone enriched cakeis preferably separated by centrifugation or filtration as describedabove with respect to separating an isoflavone enriched cake from theliquid fraction of a condensed soy molasses material.

[0055] The separated cake of aglucone isoflavone enriched materialcontains a significant amount of aglucone isoflavones therein.Preferably the cake of aglucone isoflavone enriched material contains atleast 20 mg of aglucone isoflavones per gram of cake material, on a drybasis (at least about 2% aglucone isoflavones by weight) and morepreferably contains from about 20 mg to about 50 mg of agluconeisoflavones per gram of cake material (from about 2% to about 5%aglucone isoflavones, by weight).

[0056] An aglucone isoflavone enriched material may also be producedfrom an isoflavone enriched material recovered from soy molasses, wherethe process for recovering an isoflavone enriched material from soymolasses is described above. Water is added to the recovered cake ofisoflavone enriched material to form a slurry of the isoflavone enrichedmaterial. Preferably the slurry is diluted to about 6% to about 13%solids, although a higher solids content may be used. Isoflavoneconjugates in the slurry are then converted to isoflavone glucosides bytreating the slurry under the same conditions described above withrespect to converting isoflavone conjugates to isoflavone glucosides insoy molasses. In particular, the slurry is treated at a pH of about 6 toabout 13.5, preferably about pH 9 to about pH 11, and a temperature ofabout 2° C. to about 120° C. for a period of about 15 minutes to severalhours. Most preferably the slurry is treated at a pH of about 11 and atemperature of about 5° C. to about 50° C., preferably about 35° C., fora period of about 30 minutes to about 1 hour; or at a pH of about 9 anda temperature of about 45° C. to about 75° C., preferably about 73° C.,for a period of about 4 hours to about 6 hours. If desired, anisoflavone glucoside enriched material may be separated from the slurryin a manner similar to the separation of an isoflavone enriched materialfrom soy molasses described above.

[0057] Isoflavone glucosides in the slurry are then converted toaglucone isoflavones under the same conditions described above withrespect to converting isoflavone glucosides to aglucone isoflavones in asoy molasses material. In particular, the isoflavone glucosides in theslurry are contacted with an enzyme capable of cleaving the glucosidiclinkage between the isoflavone moiety and the glucose molecule of theisoflavone glucosides under suitable pH and temperature conditions for aperiod of time sufficient to convert the isoflavone glucosides toaglucone isoflavones. Preferred enzymes, pH conditions, temperatures,and time periods are described above. An aglucone isoflavone enrichedmaterial may be separated from the slurry in a manner similar to theseparation of an isoflavone enriched material from soy molassesdescribed above.

[0058] An aglucone isoflavone enriched material may also be producedfrom an isoflavone glucoside enriched material recovered from a soymolasses material, where the process for recovering an isoflavoneglucoside enriched material from a soy molasses material is describedabove. Water is added to the recovered cake of isoflavone glucosideenriched material to form a slurry of the isoflavone glucoside enrichedmaterial. Preferably the slurry is diluted to about 6% to about 13%solids, although a higher solids content may be used. The isoflavoneglucosides in the slurry are converted to aglucone isoflavones in thesame manner described above with respect to the isoflavone glucosideenriched slurry formed from an isoflavone enriched slurry. An agluconeisoflavone enriched material may be separated from the slurry after theconversion in a manner similar to the separation of an isoflavoneenriched material from soy molasses described above.

Experimental

[0059] The present invention is illustrated in more detail by thefollowing examples. The examples are intended to be illustrative, andshould not be interpreted as limiting or otherwise restricting the scopeof the invention in any way.

[0060] As noted above, soy materials, including soy molasses, includethe genistein, daidzein, and glycitein “families” of isoflavones havingcorresponding glucoside, conjugate, and aglucone members, where thegenistein family contains the conjugates 6″-OMal genistin, 6″-OAcgenistin, the glucoside genistin, and the aglucone genistein; thedaidzein family contains the conjugates 6″-OMal daidzin, 6″-OAc daidzin,the glucoside daidzin, and the aglucone daidzein; and the glyciteinfamily includes the conjugate 6″-OMal glycitin, the glucoside glycitin,and the aglucone glycitein. In the following examples the relativeconcentrations of isoflavones are measured either as a totalconcentration of an isoflavone family, or as individual percentages ofeach isoflavone in a family of isoflavones. For example, the totalconcentration of the genistein family of isoflavones is the sum of theconcentrations of 6″-OMal genistin, 6″-OAc genistin, genistin, andgenistein, and the percentage of each of the isoflavones in thegenistein family is determined relative to the other genistein familyisoflavones: % genistin+% 6″OMal genistin+% 6″OAc genistin+%genistein=100%.

EXAMPLE 1

[0061] In a first experiment, the recovery of an isoflavone enrichedmaterial from soy molasses is examined at various concentrations of soymolasses. The total concentration of each isoflavone family is measuredin a soy molasses sample having a selected concentration, in a cakeseparated from the soy molasses sample according to the method of theinvention, and in the liquid whey from which the cake is removed by theseparation procedure.

[0062] Soy molasses is analyzed for the total concentration of all formsof isoflavones present. Samples of the soy molasses are diluted withwater to a solids content of 28% (1:2 dilution), 13.7% (1:4 dilution),and 6.6% (1:8 dilution). All samples are pH adjusted to 4.5. The treatedsamples are then centrifiged at a rate of between 3000 rpm for 30minutes to separate and produce liquid whey and cake portions from thesamples. One set of samples is centrifuged at a temperature of 0.6° C.Samples having 28% and 13.7% soy molasses solids are centrifuged at atemperature of 60° C. for comparison with samples having the sameconcentration of soy molasses solids that are separated at 0.6° C. Theresulting liquid and cake portions of the samples are analyzed for thetotal concentration of all forms of isoflavones present.

[0063] Table 1 sets forth the concentrations of isoflavones in thevarious cake and liquid fractions obtained from the previously describedtesting. The indicated totals are the totals of all forms of theparticular isoflavone including conjugates, glucoside, and agluconeforms expressed in mg of isoflavone per gram of cake or liquid fractionsolids. TABLE 1 Total Total Total Genistein Daidzein Glycitein (family)(family) (family) Sample mg/g mg/g mg/g Soy Molasses 6.1 4.8 1.0Starting Material Not separated 1:2 Dilution (28% solids) 2.8 3.0 0.6Whey separated at 0.6° C. 1:2 Dilution 16.9 10.9 1.9 Cake separated at0.6° C. 1:2 Dilution 3.8 4.0 0.8 Whey separated at 60° C. 1:2 Dilution14.1 8.2 1.5 Cake separated at 60° C. 1:4 Dilution (13.7% solids) 3.03.4 0.7 Whey separated at 0.6° C. 1:4 Dilution 18.3 11.0 2.0 Cakeseparated at 0.6° C. 1:4 Dilution 4.4 4.3 0.8 Whey separated at 60° C.1:4 Dilution 13.4 7.2 1.5 Cake separated at 60° C. 1:8 Dilution (6.6%solids) 4.3 4.5 0.9 Whey separated at 0.6° C. 1:8 Dilution 20.1 10.2 2.1Cake separated at 0.6° C.

[0064] In all separated samples, the concentration of isoflavones issignificantly higher in the cake than in soy molasses starting materialand much higher than the concentration of isoflavones in the liquid wheyfraction solids. The samples separated at 0.6° C. contained a higherconcentration of isoflavones in the cake than corresponding samplesseparated at 60° C., which had higher concentrations of isoflavones inthe whey fraction solids.

EXAMPLE 2

[0065] In another experiment, the recovery of an isoflavone glucosideenriched material from soy molasses is examined. Isoflavone conjugatesin the soy molasses are converted to isoflavone glucosides, and anisoflavone glucoside enriched cake is separated from the soy molassesmaterial. The extent of conversion is determined by the quantitativedecrease of the percentage and concentration of malonate and acetateesters of an isoflavone family coupled with a corresponding quantitativeincrease of the percentage of the glucoside of the same isoflavonefamily.

[0066] Soy molasses starting material is analyzed for concentration ofindividual isoflavone compounds. Two samples of the soy molassesmaterial are made by diluting the soy molasses with water in thefollowing ratios: 1:4(100 g of molasses+300 g water); and 1:8(50 g ofmolasses+350 g water). The pH of the samples is adjusted to 11, and thetemperature of the samples is held at 35° C. for 30 minutes. The pH ofthe samples is then adjusted to 4.5 and the temperature is adjusted to4° C. The samples are centrifuged at 10,000 rpm at 4° C. to separate themolasses samples into a cake and a liquid whey. The whey and the cakeare analyzed for concentration of individual isoflavone compounds.

[0067] Table 2 illustrates the change in the proportions between thevarious forms of isoflavones resulting from the conversion of isoflavoneconjugates to isoflavone glucosides as compared to the soy molassesstarting material. Isoflavone concentrations are indicated as parts permillion(ppm) within the sample, and as percentages of the total amountof the particular isoflavone (the total of the conjugate, glucoside, andaglucone forms) within the liquid or cake portion. TABLE 2 6″- 6″- 6″-6″- 6″- OMAL- OAC- OMAL- OAC- OMAL- Sample Genistin Genistin GenistinGenistein Daidzin Daidzin Daidzin Daidzein Glycitin Glycitin GlyciteinSoy Molasses ppm 4678 1329 0 88 3533 928 210 84 500 105 360 % isoflavone77 22 0 1 74 20 4 2 52 11 37 1:4 Dilution Whey ppm 2221 17 0 30 2652 17929 21 341 28 0 % isoflavone 98 1 0 1 92 6 1 1 92 8 0 1:4 Dilution Cakeppm 28621 68 0 261 16133 192 0 232 1442 0 66 % isoflavone 99 0 0 1 97 10 1 96 0 4 1:8 Dilution Whey ppm 2852 24 0 36 3356 187 0 27 406 28 0 %isoflavone 98 1 0 1 94 5 0 1 94 6 0 1:8 Dilution Cake ppm 27517 101 0272 12617 138 0 245 1146 0 0 % isoflavone 99 0 0 1 97 1 0 2 100 0 0

[0068] In all samples subjected to conditions for conversion ofisoflavone conjugates to isoflavone glucosides the percentage ofisoflavone glucosides in both cake and liquid portions is significantlyhigher than in the corresponding unconverted soy molasses sample, andthe percentage of corresponding isoflavone conjugates in the samples issignificantly lower, demonstrating that a large portion of isoflavoneconjugates are converted to their glucoside form. Furthermore, uponseparation a large proportion of the glucoside isoflavones are separatedin the cake to form an isoflavone glucoside enriched material, as can beseen from the relative concentrations of the soy molasses startingmaterial and the whey and cake portions of each sample.

EXAMPLE 3

[0069] In another experiment, the conversion of isoflavones to agluconeisoflavones in soy molasses is examined. Isoflavone conjugates in thesoy molasses are converted to isoflavone glucosides, and the isoflavoneglucosides are then converted to aglucone isoflavones. The extent ofconversion of the isoflavone glucosides to aglucone isoflavones isdetermined by the quantitative decrease of the concentration of theglucoside of an isoflavone family coupled with a correspondingquantitative increase of the percentage of the aglucone of the sameisoflavone family.

[0070] Soy molasses starting material is diluted 1:4 with water and isanalyzed for concentration of individual isoflavone compounds. The pH ofthe molasses is then adjusted to 11. The soy molasses is held at roomtemperature for 1 hour to produce a glucoside enriched soy molassesmaterial. The glucoside enriched soy molasses material is analyzed forconcentration of individual isoflavone compounds. Four samples areprepared from the glucoside enriched soy molasses material after the pHof the material is adjusted to 4.5. Each sample is inoculated with anenzyme, where the following enzymes are added to the samples,respectively, at 10% by weight of the molasses solids in each sample:G-Zyme 990, Biopectinase 100L, Lactase 50,000, and Alpha-Gal 600L. Thesamples are then treated at 50° C. for 6 hours to form an agluconeisoflavone enriched soy molasses material. The aglucone isoflavoneenriched soy molasses is then analyzed for isoflavone content.

[0071] Table 3 illustrates the distribution between the various forms ofisoflavones resulting from the previously described testing. Isoflavoneconcentrations are indicated as parts per million(ppm) within thesample, and as percentages of the total amount of the particularisoflavone (the total of the conjugate, glucoside, and aglucone forms).TABLE 3 6″- 6″- 6″- 6″- 6″- OMAL- OAC- OMAL- OAC- OMAL- Sample GenistinGenistin Genistin Genistein Daidzin Daidzin Daidzin Daidzein GlycitinGlycitin Glycitein Soy Molasses ppm 4678 1329 0 88 3533 928 210 84 500105 360 % isoflavone 77 22 0 1 74 20 4 2 52 11 37 Glucoside rich soymolasses ppm 6763 0 0 104 4377 0 0 43 433 0 0 % isoflavone 98 0 0 2 99 00 1 100 0 0 G-Zyme 990, 10% ppm 3903 0 0 1993 840 0 82 2331 346 0 114 %isoflavone 66 0 0 44 27 0 2 71 75 0 25 Biopectinase 100 L, 10% ppm 28650 0 2919 541 0 94 2701 195 0 237 % isoflavone 50 0 0 50 16 3 81 45 0 55Lactase 50,000, 10% ppm 0 0 0 4601 0 0 92 2875 0 0 366 % isoflavone 0 00 100 0 0 3 97 0 0 100 Alpha-Gal 600 L, 10% ppm 28 0 0 4566 0 0 89 28820 0 356 % isoflavone 1 0 0 99 0 0 3 97 0 0 100

[0072] The aglucone isoflavone content of the enzymatically treatedsamples is significantly higher than the soy molasses and the glucosideenriched soy molasses material, indicating that the enzymatic treatmentconverted substantial amounts of glucoside isoflavones to agluconeisoflavones. Selection of the proper enzyme, enzyme concentration, pHand temperature for the conversion permits conversion of substantiallyall of the isoflavone glucosides to aglucone isoflavones, asdemonstrated by the isoflavone distribution in the Lactase 50,000 andAlpha-Gal 600L samples.

EXAMPLE 4

[0073] In a final experiment, the isoflavone content of an isoflavoneenriched material, an isoflavone glucoside enriched material, and anaglucone isoflavone enriched material is examined and the distributionof the isoflavones in the materials is compared. Soy molasses is dilutedto a 1:4 ratio with water. A sample of the diluted soy molasses isadjusted to a pH of 4.5, is chilled to a temperature of 0.6° C. in anice bath for 30 minutes, and is centrifuged at a rate of 3000 rpm for 30minutes to separate a cake of isoflavone enriched material. Theremaining diluted soy molasses is then adjusted to a pH of 11 withsodium hydroxide and is treated at 50° C. for 1 hour to convertisoflavone conjugates in the molasses to isoflavone glucosides. A sampleof the glucoside enriched molasses is adjusted to a pH of 4.5, ischilled to a temperature of 0.6° C. in an icebath for 30 minutes, and iscentrifuged at a rate of 3000 rpm for 30 minutes to separate a cake ofisoflavone glucoside enriched material. The remaining isoflavoneglucoside enriched soy molasses material is adjusted to pH 4.5, and theenzyme G-Zyme 990 is added to the material at a concentration of 2.6 genzyme/100 g of molasses material. The enzyme and the isoflavoneglucoside enriched soy molasses material are then treated at 50° C. for18 to 20 hours to convert the isoflavone glucosides to agluconeisoflavones. A sample of the aglucone isoflavone enriched soy molassesmaterial is chilled to a temperature of 0.6° C. in an icebath for 30minutes, and is centrifuged at a rate of 3000 rpm for 30 minutes toseparate a cake of aglucone isoflavone enriched material. The recoveredcakes of isoflavone enriched material, isoflavone glucoside enrichedmaterial, and aglucone isoflavone enriched material are then analyzedfor isoflavone content.

[0074] Table 4 below shows the distribution of the isoflavones in thecakes of isoflavone enriched material, isoflavone glucoside enrichedmaterial, and aglucone isoflavone enriched material. Isoflavonedistribution is indicated as percentages of the total amount of theparticular isoflavone (the total of the conjugate, glucoside, andaglucone forms). TABLE 4 6″- 6″- 6″- 6″- 6″- OMAL- OAC- OMAL- OAC- OMAL-Sample Genistin Genistin Genistin Genistein Daidzin Daidzin DaidzinDaidzein Glycitin Glycitin Glycitein Isoflavone rich material %isoflavone 83 16 0 1 81 12 5 1 40 8 52 Glucoside rich material %isoflavone 99 0 0 1 99 0 0 1 95 0 5 Aglucone rich material % isoflavone3 0 0 97 0 0 0 100 54 18 28

[0075] The effectiveness of the conversion steps can be seen in theisoflavone distribution of the materials. The isoflavone glucosideenriched material contains significantly higher amounts of isoflavoneglucosides than the isoflavone enriched material and the agluconeisoflavone material, having an isoflavone content which is comprised ofalmost entirely isoflavone glucosides. The aglucone isoflavone enrichedmaterial contains significantly higher amounts of aglucone isoflavonesthan the isoflavone glucoside enriched material and the isoflavoneenriched material, having an isoflavone content which is comprised ofalmost entirely aglucone isoflavones.

[0076] In the above examples, all percentages indicated for6″-OMal-genistin, 6″-OAc-genistin, 6″-OMal-daidzin, 6″-OAc-daidzin,glycitin, 6″-OMal-glycitin, and glycitein are calculated values. Thefollowing is a description of a method for quantifying isoflavones insoy products. The isoflavones are extracted from soy products by mixing0.75 gram of sample (spray dried or finely ground powder) with 50 ml of80/20 methanol/water solvent. The mixture is shaken for 2 hours at roomtemperature with an orbital shaker. After 2 hours, the remainingundissolved materials are removed by filtration through Whatman No. 42filter paper. Five ml of the filtrate are diluted with 4 ml of water and1 ml of methanol.

[0077] The extracted isoflavones are separated by HPLC (High PerformanceLiquid Chromatography)using a Hewlett Packard C18 Hypersil reverse phasecolumn. The isoflavones are injected onto the column and eluted with asolvent gradient starting with 88% methanol, 10% water, and 2% glacialacetic acid and ending with 98% methanol and 2% glacial acetic acid. Ata flow rate of 0.4 ml/min, all the isoflavones-genistin,6″-0-acetylgenistin, 6″-0-malonylgenistin, genistein, daidzin,6″-0-acetyldaidzin, 6″-0-malonyldaidzin, daidzin, glycitin and itsderivatives and glycitein—are clearly resolved. Peak detection is by UVabsorbence at 260 mm. Identification of the peaks was performed byHPLC-mass spectrometer.

[0078] Quantification is achieved by using pure standards (genistin,genistein, daidzin and daidzein) obtained from Indofine ChemicalCompany, Sommerville, N.J. Response factors (integratedarea/concentration) are calculated for each of the above compounds andare used to quantitate unknown samples. For the conjugated forms forwhich no pure standards are available, response factors are assumed tobe that of the parent molecule but corrected for molecular weightdifference. The response factor for glycitin is assumed to be that forgenistin corrected for molecular weight difference. This method providesthe quantities of each individual isoflavone. For convenience, totalgenistein, total daidzein and total glycitein can be calculated, andrepresent the aggregate weight of these compounds if all the conjugatedforms are converted to their respective unconjugated forms. These totalscan also be measured directly by a method using acid hydrolysis toconvert the conjugated forms.

[0079] The foregoing are merely preferred embodiments of the invention.Various changes and alterations can be made without departing from thespirit and broader aspects thereof as set forth in the appended claims,which are to be interpreted in accordance with the principals of patentlaw including the Doctrine of Equivalents.

What is claimed is:
 1. A health supplement comprising a solid materialderived from a soy molasses material, wherein said solid materialcontains at least two isoflavones.
 2. The health supplement of claim 1wherein at least one of said isoflavones is an aglucone isoflavone. 3.The health supplement of claim 2 wherein said aglucone isoflavone isgenistein.
 4. The health supplement of claim 2 wherein said agluconeisoflavone is daidzein.
 5. The health supplement of claim 2 wherein saidaglucone isoflavone is glycitein.
 6. The health supplement of claim 2wherein at least two of said isoflavones are aglucone isoflavones. 7.The health supplement of claim 6 wherein said aglucone isoflavones aregenistein and daidzein.
 8. The health supplement of claim 6 wherein saidaglucone isoflavones are genistein and glycitein.
 9. The healthsupplement of claim 6 wherein said aglucone isoflavones are daidzein andglycitein.
 10. The health supplement of claim 2 containing genistein,daidzein, and glycitein.
 11. The health supplement of claim 1 wherein atleast one of said isoflavones is an isoflavone glucoside.
 12. The healthsupplement of claim 11 wherein said isoflavone glucoside is genistin.13. The health supplement of claim 11 wherein said isoflavone glucosideis daidzin.
 14. The health supplement of claim 11 wherein saidisoflavone glucoside is glycitin.
 15. The health supplement of claim 11wherein at least two of said isoflavones are isoflavone glucosides. 16.The health supplement of claim 15 wherein said isoflavone glucosides aregenistin and daidzin.
 17. The health supplement of claim 16 wherein saidisoflavone glucosides are genistin and glycitin.
 18. The healthsupplement of claim 16 wherein said isoflavone glucosides are daidzinand glycitin.
 19. The health supplement of claim 16 containing genistin,daidzin, and glycitin.
 20. The health supplement of claim 1 wherein atleast one of said isoflavones is an isoflavone glucoside conjugate. 21.The health supplement of claim 20 wherein said isoflavone glucosideconjugate is 6″-O-acetyl genistin.
 22. The health supplement of claim 20wherein said isoflavone glucoside conjugate is 6″-O-malonyl genistin.23. The health supplement of claim 20 wherein said isoflavone glucosideconjugate is 6″-O-acetyl daidzin.
 24. The health supplement of claim 20wherein said isoflavone glucoside conjugate is 6″-O-malonyl daidzin. 25.The health supplement of claim 20 wherein said isoflavone glucosideconjugate is 6″-O-malonyl glycitin.
 26. The health supplement of claim 1wherein at least one of said isoflavones is an aglucone isoflavone andat least one of said isoflavones is an isoflavone glucoside.
 27. Thehealth supplement of claim 26 wherein said aglucone isoflavone isgenistein.
 28. The health supplement of claim 27 wherein said isoflavoneglucoside is genistin.
 29. The health supplement of claim 27 whereinsaid isoflavone glucoside is daidzin.
 30. The health supplement of claim27 wherein said isoflavone glucoside is glycitin.
 31. The healthsupplement of claim 27 containing at least two isoflavone glucosides.32. The health supplement of claim 31 wherein said isoflavone glucosidesare genistin and daidzin.
 33. The health supplement of claim 31 whereinsaid isoflavone glucosides are genistin and glycitin.
 34. The healthsupplement of claim 31 wherein said isoflavone glucosides are daidzinand glycitin.
 35. The health supplement of claim 31 containing genistin,daidzin, and glycitin.
 36. The health supplement of claim 26 whereinsaid aglucone isoflavone is daidzein.
 37. The health supplement of claim36 wherein said isoflavone glucoside is genistin.
 38. The healthsupplement of claim 36 wherein said isoflavone glucoside is daidzin. 39.The health supplement of claim 36 wherein said isoflavone glucoside isglycitin.
 40. The health supplement of claim 36 containing at least twoisoflavone glucosides.
 41. The health supplement of claim 40 whereinsaid isoflavone glucosides are genistin and daidzin.
 42. The healthsupplement of claim 40 wherein said isoflavone glucosides are genistinand glycitin.
 43. The health supplement of claim 40 wherein saidisoflavone glucosides are daidzin and glycitin.
 44. The healthsupplement of claim 40 containing genistin, daidzin, and glycitin. 45.The health supplement of claim 26 wherein said aglucone isoflavone isglycitein.
 46. The health supplement of claim 46 wherein said isoflavoneglucoside is genistin.
 47. The health supplement of claim 46 whereinsaid isoflavone glucoside is daidzin.
 48. The health supplement of claim46 wherein said isoflavone glucoside is glycitin.
 49. The healthsupplement of claim 46 containing at least two isoflavone glucosides.50. The health supplement of claim 49 wherein said isoflavone glucosidesare genistin and daidzin.
 51. The health supplement of claim 49 whereinsaid isoflavone glucosides are genistin and glycitin.
 52. The healthsupplement of claim 49 wherein said isoflavone glucosides are daidzinand glycitin.
 53. The health supplement of claim 49 containing genistin,daidzin, and glycitin.
 54. The health supplement of claim 26 containingat least two aglucone isoflavones
 55. The health supplement of claim 54wherein said aglucone isoflavones are genistein and daidzein.
 56. Thehealth supplement of claim 55 wherein said isoflavone glucoside isgenistin.
 57. The health supplement of claim 55 wherein said isoflavoneglucoside is daidzin.
 58. The health supplement of claim 55 wherein saidisoflavone glucoside is glycitin.
 59. The health supplement of claim 55containing at least two isoflavone glucosides.
 60. The health supplementof claim 59 wherein said isoflavone glucosides are genistin and daidzin.61. The health supplement of claim 59 wherein said isoflavone glucosidesare genistin and glycitin.
 62. The health supplement of claim 59 whereinsaid isoflavone glucosides are daidzin and glycitin.
 63. The healthsupplement of claim 59 containing genistin, daidzin, and glycitin. 64.The health supplement of claim 54 wherein said aglucone isoflavones aregenistein and glycitein.
 65. The health supplement of claim 64 whereinsaid isoflavone glucoside is genistin.
 66. The health supplement ofclaim 64 wherein said isoflavone glucoside is daidzin.
 67. The healthsupplement of claim 64 wherein said isoflavone glucoside is glycitin.68. The health supplement of claim 64 containing at least two isoflavoneglucosides.
 69. The health supplement of claim 68 wherein saidisoflavone glucosides are genistin and daidzin.
 70. The healthsupplement of claim 68 wherein said isoflavone glucosides are genistinand glycitin.
 71. The health supplement of claim 68 wherein saidisoflavone glucosides are daidzin and glycitin.
 72. The healthsupplement of claim 68 containing genistin, daidzin, and glycitin. 73.The health supplement of claim 54 wherein said aglucone isoflavones aredaidzein and glycitein.
 74. The health supplement of claim 73 whereinsaid isoflavone glucoside is genistin.
 75. The health supplement ofclaim 73 wherein said isoflavone glucoside is daidzin.
 76. The healthsupplement of claim 73 wherein said isoflavone glucoside is glycitin.77. The health supplement of claim 73 containing at least two isoflavoneglucosides.
 78. The health supplement of claim 77 wherein saidisoflavone glucosides are genistin and daidzin.
 79. The healthsupplement of claim 77 wherein said isoflavone glucosides are genistinand glycitin.
 80. The health supplement of claim 77 wherein saidisoflavone glucosides are daidzin and glycitin.
 81. The healthsupplement of claim 77 containing genistin, daidzin, and glycitin. 82.The health supplement of claim 1 containing genistein, daidzein,glycitein, genistin, daidzin, and glycitin.